US11193136B2 - Cellulose synthase inhibitors and mutant plants - Google Patents
Cellulose synthase inhibitors and mutant plants Download PDFInfo
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- US11193136B2 US11193136B2 US16/323,707 US201716323707A US11193136B2 US 11193136 B2 US11193136 B2 US 11193136B2 US 201716323707 A US201716323707 A US 201716323707A US 11193136 B2 US11193136 B2 US 11193136B2
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- 0 [1*]C1=C([2*])C=C(C2CC(C3=C(OC)C=CC=C3)N3N=C([3*])N=C3N2)C=C1 Chemical compound [1*]C1=C([2*])C=C(C2CC(C3=C(OC)C=CC=C3)N3N=C([3*])N=C3N2)C=C1 0.000 description 6
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
- C12N9/1059—Cellulose synthases (2.4.1.12; 2.4.1.29)
Definitions
- the present invention relates to specific inhibitors of the cellulose synthase subunits 1 and 3 activity in plants, useful as a herbicide.
- the invention relates to mutant plants which are tolerant to the identified inhibitors.
- Specific mutant alleles of CESA1 and CESA3 genes can be used to obtain resistance in a plant when the inhibitors are used as herbicide.
- Plant cells walls are essential for the plant rigidity and strength, but they protect the plant also against environmental stress.
- Cellulose is one of the major compounds of the plant cell wall.
- Cellulose is a hydrogen bonded beta-1,4-linked glucan microfibril, and is synthesized by large multimeric cellulose synthase complexes (CSC).
- CSCs can be divided in a primary cell wall CSC and a secondary cell wall CSC (Endler and Person, 2011).
- the CSC is a large, hexameric rosette like structure, comprising six globular protein complexes (Kimura et al., 1999).
- Each of those complexes is holding 6 cellulose synthase subunits (CESAs); the CESAs are believed to be the catalytic subunits of the complex.
- CESAs cellulose synthase subunits
- At least three different CESAs are present in a complex (Festucci-Buselli et al., 2007; Lei et al., 2012); the CSC of the primary cell wall synthesis is composed of CESA 1, 3 and 6 (or 6-like proteins CESA2,5 and 9), the CSC of the secondary cell wall synthesis is composed of CESA 4, 7 and 8 (Lei et al., 2012).
- cellulose synthase is an interesting target for herbicides, as cellulose synthase inhibitors are expected to be efficient in herbicidal activity without major negative effects on animal life forms.
- cellulose biosynthesis inhibitors such as dichlobenil, isoxaben, quinoxyphen and flupoxam have indeed been developed as herbicides. The mode of action of those herbicides has been studied, and the four herbicides work at a different level (Brabham and DeBolt, 2013).
- FIG. 1 Structure of the different herbicide variants, and their effect on the growth of wild type Arabidopsis (Col-0) and a resistant mutant CESA1 A1018V (indicated as cesa1 7I ).
- FIG. 2 Left panel: inhibitory effect on root growth of the compound C17(7693622) at different concentrations. Right panel: analysis of cell death in the root of — Arabidopsis , caused by increasing concentrations of the compound C17(7693622).
- FIG. 3 Identification of mutants, resistant against compound C17(7693622).
- A. Fine mapping of the mutation cesa1 7I ( CESA1 A1080V ) identified in CESA1.
- B. Fine mapping of the mutation cesa3 2c ( CESA3 S983F ) identified in CESA3.
- FIG. 4 Sequence alignment of CESA (A) ( A.thaliana (SEQ ID NO:3); G.max (SEQ ID NO:4); F.vesca (SEQ ID NO:5); V.vinifera (SEQ ID NO:6); S.lycopersicum (SEQ ID NO:7); Z.mays (SEQ ID NO:8); and O.sativa (SEQ ID NO:9)); and CESA3 (B) ( A.thaliana (SEQ ID NO:10); G.max (SEQ ID NO:11); F.vesca (SEQ ID NO:12); V.vinifera (SEQ ID NO:13); S.lycopersicum (SEQ ID NO:14); Z.mays (SEQ ID NO:15); and O.sativa (SEQ ID NO:16)).
- A thaliana
- G.max SEQ ID NO:4
- F.vesca SEQ ID NO:5
- V.vinifera SEQ ID NO:6
- Sequences were aligned with a multiple sequence alignment programme (world wide web at genome.jp/tools/clustalw/) using CLUSTALW algorithms.
- Protein database accession numbers are: CESA1 A.thaliana -NP_194967; CESA1 G.max -XP_003522623; CESA1 F.vesca -XP_004291468; CESA1 V.vinifera -XP_002282575; CESA1 S.lycopersicum -XP_004245031; CESA1 Z.mays -NP_001104954; CESA1 O.sativa -NP_001054788; CESA3 A.thaliana -NP_196136; CESA3 G.max -XP_003540527; CESA3 F.vesca -XP_004306536; CESA3 V.vinifera -XP_002278997; CESA3
- FIG. 5 Sensitivity of Isoxaben resistant mutants to the compound C17(7693622), proving the different working mechanism of compound C17(7693622).
- FIG. 6 Use of the combination of compound C17(7693622) as inhibiting compound and the CESA3 resistance gene as transformation marker: plants carrying the resistant gene can easily be distinguished from the non-transformed control.
- FIG. 7 The application of compound C17 results in a brittle cell wall.
- each of the following terms has the meaning associated with it in this section.
- the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
- an element means one element or more than one element.
- “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
- abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
- observable or detectable characteristic e.g., age, treatment, time of day, etc.
- Characteristics which are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.
- the invention provides a mutant CESA1 or mutant CESA3 gene wherein said mutation encodes for a mutant CESA1 or CESA3 protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 P1010L , CESA1 G1013R , CESA1 G1013E , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T , CESA1 V1023T CESA3 S983F and CESA3 S1037F .
- the invention provides a mutant CESA1 or mutant CESA3 gene wherein said mutation encodes for a mutant CESA1 or CESA3 protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T CESA1 L1023T and CESA1 V1023T .
- the invention provides a plant having a mutation in the CESA1 or CESA3 gene wherein said mutation encodes for a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R CESA1 P1010L , CESA1 G1013R , CESA1 G1013E , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T , CESA1 V1023T , CESA3 S983F and CESA3 S1037F .
- the invention provides a plant having a mutation in the CESA1 or CESA3 gene wherein said mutation encodes for a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T and CESA1 V1023T .
- the plant has a mutation in at least one CESA1 or CESA3 allele said mutation encodes for a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 P1010L , CESA1 G1013R , CESA1 G1013E , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T , CESA1 V1023T , CESA3 S983F and CESA3 S1037F .
- the plant has a mutation in at least one CESA1 or CESA3 allele said mutation encodes for a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 A1018V CESA1 S1018V , CESA1 A1023T , CESA1 L1023T and CESA1 V1023T .
- the plant has a mutation in at least two CESA1 or CESA3 alleles said mutation encodes for a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 P1010L , CESA1 G1013R , CESA1 G1013E , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T , CESA1 V1023T , CESA3 S983F and CESA3 S1037F .
- the plant has a mutation in at least two CESA1 or CESA3 alleles said mutation encodes for a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T and CESA1 V1023T .
- the plant has a mutation in all CESA1 or CESA3 alleles said mutation encodes for a mutant protein selected from the group consisting of CESA1 V2971M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K94512 , CESA1 P1010L , CESA1 G1013R , CESA1 G1013E , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T , CESA1 V1023T CESA3 S983F and CESA3 S1037F .
- the plant has a mutation in all CESA1 or CESA3 alleles said mutation encodes for a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T and CESA1 V1023T .
- CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease with an engineered crRNA/tracr RNA
- ZFNs Zinc Finger Nucleases
- TALENS Transcription Activator-Like Effector Nucleases
- CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease with an engineered crRNA/tracr RNA
- U.S. Patent Publication No. 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and WO 2007/014275 the disclosures of which are incorporated by reference in their entireties for all purposes.
- U. 520080182332 describes the use of non-canonical zinc finger nucleases (ZFNs) for targeted modification of plant genomes and
- U.S. Patent Publication No. 20090205083 describes ZFN-mediated targeted modification of a plant specific genomic locus.
- Current methods for targeted insertion of exogenous DNA typically involve co-transformation of plant tissue with a donor DNA polynucleotide containing at least one transgene and a site specific nuclease (e.g. ZFN) which is designed to bind and cleave a specific genomic locus of an actively transcribed coding sequence.
- ZFNs site specific nuclease
- zinc fingers defines regions of amino acid sequence within a DNA binding protein binding domain whose structure is stabilized through coordination of a zinc ion.
- a “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
- the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
- Zinc finger binding domains can be “engineered” to bind to a predetermined nucleotide sequence.
- Non-limiting examples of methods for engineering zinc finger proteins are design and selection.
- a designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria.
- Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261 and 6,794,136; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
- a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence.
- a single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference herein in its entirety.
- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- Cas CRISPR Associated nuclease system.
- a “CRISPR DNA binding domain” is a short stranded RNA molecule that acting in concert with the CAS enzyme can selectively recognize, bind, and cleave genomic DNA.
- the CRISPR/Cas system can be engineered to create a double-stranded break (DSB) at a desired target in a genome, and repair of the DSB can be influenced by the use of repair inhibitors to cause an increase in error prone repair. See, e.g., Jinek et al (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471, and David Segal, (2013) eLife 2:e00563).
- Zinc finger, CRISPR and TALE binding domains can be “engineered” to bind to a predetermined nucleotide sequence such as a specific site in the CESA1 or CESA3 gene, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger.
- a predetermined nucleotide sequence such as a specific site in the CESA1 or CESA3 gene
- Yet another possibility is the use of targeted nucleotide editing of DNA using hybrid vertebrate and bacterial immune systems components.
- Nuclease-deficient type II CRISPR/Cas9 and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 can be engineered to form a synthetic complex (Target-AID) to perform highly efficient target-specific mutagenesis (see Nishida K.
- TALEs can be “engineered” to bind to a predetermined nucleotide sequence, for example by engineering of the amino acids involved in DNA binding (the repeat variable di-residue or RVD region). Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring.
- Non-limiting examples of methods for engineering DNA-binding proteins are design and selection.
- a designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos.
- a “selected” zinc finger protein, CRISPR or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO 02/099084 and U.S. Publication Nos. 20110301073, 20110239315 and 20119145940.
- mutagenesis such as ethyl methanesulfonate (EMS)-induced mutagenesis and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous CESA1 or CESA3 gene has been mutated.
- EMS ethyl methanesulfonate
- fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous CESA1 or CESA3 gene has been mutated.
- the invention encompasses still additional methods for mutating one or more CESA1 and/or CESA3 alleles.
- methods for altering or mutating a genomic nucleotide sequence in a plant include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides and recombinogenic oligonucleotide bases.
- Such vectors and methods of use are known in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, each of which are herein incorporated by reference.
- a “chimeric gene” or “chimeric construct” is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence.
- the regulatory nucleic acid sequence of the chimeric gene is not operatively linked to the associated nucleic acid sequence as found in nature.
- a “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells.
- the nucleic acid molecule For expression in plants, the nucleic acid molecule must be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.
- operably linked refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
- a “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ.
- plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
- plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
- Plants that are particularly useful in the methods of the invention include in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp.
- Avena sativa e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida
- Averrhoa carambola e.g. Bambusa sp.
- Benincasa hispida Bertholletia excelsea
- Beta vulgaris Brassica spp.
- Brassica napus e.g. Brassica napus, Brassica rapa ssp.
- the invention provides a plant transformation marker system, comprising a herbicide with the structure
- R1 is a halogen
- R2 is H or a halogen
- R3 is H or —N (CH 3 ) 2 as a herbicide to inhibit cellulose biosynthesis.
- the invention provides a method to transform plants, said method comprising (1) using a vector comprising a mutant CESA1 or CESA3 gene encoding a mutant protein selected from the group consisting of CESA1 V297M , CESA1 S307L , CESA1 L872F , CESA1 S892N , CESA1 G892N , CESA1 K945R , CESA1 P10L , CESA1 G1013R , CESA1 G1013E , CESA1 A1018V , CESA1 S1018V , CESA1 A1023T , CESA1 L1023T , CESA1 V1023T , CESA3 S983F and CESA3 S1037F , and (2) selecting the transformants using a compound as herbicide according to the invention.
- said compound is incorporated in the medium.
- the vector comprising a mutant CESA1 or CESA3 gene can control the expression of the gene under its own CESA1 or CESA3 promoter or alternatively the CESA1 or CESA3 gene can be brought under control of a heterologous promoter.
- C17 (5-(4-chlorophenyl)-7-(2-methoxyphenyl)-1,5,6,7-tetrahydro-[1,2,4]triazolo[1,5-a]pyrimidine; ChemDiv, Catalogue #: 7693622), a synthetic molecule, was identified from a chemical screen as a ploidy-inducing compound in Arabidopsis protoplasts.
- Arabidopsis seeds were plated on half MS medium (Murashige, T. and F. Skoog, 1962) in the presence or absence of 200 nM C17 or its analogues.
- C17-treated plants displayed severe growth inhibition accompanied by radial swelling of the root tip ( FIG. 1 ).
- EMS ethylmethanesulphonate
- CESA1/CESA3 homologs from 7 species revealed that 9 mutated amino acids are invariant ( FIG. 4 ).
- CESA1 and CESA3 are two crucial components of cellulose synthase complexes (CSCs) that catalyse the deposition of cellulose, of which the dysfunction results in swollen roots, defective cell elongation, and cell death. Applying C17 to wild type plant mimics these deformities, strengthening the observation that C17 is a cellulose synthase inhibitor.
- the C17 compound differs completely from any known cellulose inhibitor.
- Isoxaben is a typical and potent cellulose synthase inhibitor, for which resistant mutants (ixr1-1, ixr1-2, and ixr2-1) have been described, corresponding to mutant alleles of CESA3 and CESA6.
- ixr1-1, ixr1-2, and ixr2-1 are still sensitive towards C17 ( FIG. 5 ), illustrating that C17 and isoxaben operate differently.
- CESA3 S983F cesa3 2c mutant allele
- C17 treatment resulted in the depletion of the CESA complex from the plasma membrane of wild-type root cells with a dramatic drop after 10 to 15 min of C17 application.
- the cellulose synthesized by the CESA1/CESA3 complex is a primary cell wall component, it was expected that C17-treated plants would display a weaker cell wall, which can be visualized by the uptake of propidium iodide (PI) following the application of a gentle pressure on the root.
- PI propidium iodide
- the ixr1-1 (collection number CS6201), ixr1-2 (CS6202) and ixr2-1 (CS6203) mutants were acquired from the ABRC. Five-day-old Arabidopsis seedlings were transferred to half MS medium with or without 200 nM C17. After 3 day, the plants were photographed.
- CESA1 and CESA3 Protein amino acid sequences of CESA1 and CESA3 from 7 species, extracted from the GenBank database, were aligned using CLUSTALW. Sequence data can be found under the following accession numbers: CESA1 A.thaliana (NP_194967), CESA1 G.max (XP_003522623), CESA1 F.vesca (XP_004291468), CESA1 V.vinifera (XP_002282575), CESA1 S.lycopersicum (XP_004245031), CESA1 Z.mays (NP_001104954), CESA1 O.sativa (NP_001054788), CESA3 A.thaliana (NP_196136), CESA3 G.max (XP_003540527), CESA3 F.vesca (XP_004306536), CESA3 V.vinifera (XP_002278997), CESA3 S
- CESA3 2C sequences were amplified from cDNA of CESA3 2C mutant plants by PCR using the following primer pairs (CESA3_ATTB1: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCA TGGAATCCGAAGGAGAAACCGCG (SEQ ID NO: 1); CESA3_ATTB2: GGGGACCACTTT GTACAAGAAAGCTGGGTGTCGCTTC-TCAACAGTTGATTCC SEQ ID NO: 2).
- the resulting fragments were created with the Pfu DNA polymerase kit (Promega) and were cloned into a pDONR221 entry vector by BP recombination cloning and subsequently transferred into the modified pGWB2 destination vector in which 35S promoter was replace by CESA3 promoter.
- Arabidopsis plants (cesa3 je5 ) were transformed using the floral-dip method as described (Clough and Bent, 1998). T1 seeds were germinated on MS medium supplemented with 2 ⁇ M C17 or 25 mg/l hygromycin to screen for transformants.
Abstract
Description
-
- a. a plant expressible promoter,
- b. a mutant CESA1 or CESA3 gene encoding a mutant protein selected from the group consisting of CESA1V297M, CESA1S307L, CESA1L872F, CESA1S892N, CESA1G892N, CESA1K945R, CESA1P1010L, CESA1G1013R, CESA1G1013E, CESA1A1018V, CESA1S1018V, CESA1A1023T, CESA1L1023T, CESA1V1023T, CESA3S983F and CESA3S1037F,
- c. a 3′ end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
-
- a. a plant expressible promoter,
- b. a mutant CESA1 or CESA3 gene encoding a mutant protein selected from the group consisting of CESA1V297M, CESA1S307L, CESA1L872F, CESA1S892N, CESA1G892N, CESA1K945R, CESA1A1018V, CESA1S1018V, CESA1A1023T, CESA1L1023T and CESA1V1023T,
- c. a 3′ end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
-
- a. a plant expressible promoter,
- b. a mutant CESA1 or CESA3 gene encoding a mutant protein selected from the group consisting of CESA1V297M, CESA1S307L, CESA1L872F, CESA1S892N, CESA1G892N, CESA1K945R, CESA1P010L, CESA1G1013R, CESA1G1013E, CESA1A1018V, CESA1S1018V, CESA1A1023T, CESA1L1023T, CESA1V1023T, CESA3S983F and CESA3S1037F,
- c. a 3′ end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
-
- a. a plant expressible promoter,
- b. a mutant CESA1 or CESA3 gene encoding a mutant protein selected from the group consisting of CESA1V297M, CESA1S307L, CESA1L872F, CESA1S892N, CESA1G892N, CESA1K945R, CESA1A1018V, CESA1S1018V, CESA1A1023T, CESA1L1023T and CESA1V1023T,
- c. a 3′ end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
-
- wherein R1 is a halogen, R2 is H or a halogen and R3 is H or —N (CH3)2 and a herbicide resistance gene, which encodes a mutant protein of cellulose synthase subunit 1 (CESA1) or cellulose synthase subunit 3 (CESA3) selected from the group consisting of CESA1V297M, CESA1S307L, CESA1L872F, CESA1S892N, CESA1G892N, CESA1K945R, CESA1P1010L, CESA1G1013R, CESA1G1013E, CESA1A1018V, CESA1S1018V, CESA1A1023T, CESA1L1023T, CESA1V1023T, CESA3S983F and CESA3S1037F.
-
- wherein R1 is a Cl, R2 is H or a halogen and R3 is H and a herbicide resistance gene, which encodes a mutant protein of cellulose synthase subunit 1 (CESA1) or cellulose synthase subunit 3 (CESA3) selected from the group consisting of CESA1V297M, CESA1S307L, CESA1L872F, CESA1S82N, CESA1G892N, CESA1K945R, CESA1P1010L, CESA1G1013R, CESA1G1013E, CESA1A1018V, CESA1S1018V, CESA1A1023T, CESA1L1023T, CESA1V1023T, CESA3S983F and CESA3S1037F.
-
- wherein R1 is a halogen, R2 is H or a halogen and R3 is H or —N (CH3)2 as a herbicide.
wherein R1 is a halogen, R2 is H or a halogen and R3 is H or —N (CH3)2 as a herbicide to inhibit cellulose biosynthesis.
-
- a. a plant expressible promoter,
- b. a mutant CESA1 or CESA3 gene encoding a mutant protein selected from the group consisting of CESA1V297M, CESA1S307L, CESA1L872F, CESA1S892N, CESA1G892N, CESA1K945R, CESA1P1010L, CESA1G1013R, CESA1G1013E, CESA1A1018V, CESA1S1018V, CESA1A1023T, CESA1L1023T, CESA1V1023T, CESA3S983F and CESA3S1037F,
- c. a 3′ end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
- Austin, R. S., Vidaurre, D., Stamatiou, G., Breit, R., Provart, N. J., Bonetta, D., Zhang, J., Fung, P., Gong, Y., Wang, P. W., McCourt, P., and Guttman, D. S. 2011. Next-generation mapping of Arabidopsis genes. Plant J 67, 715-725.
- Brabham, C and DeBolt, S. 2013. Chemical genetics to examine cellulose biosynthesis. Frontiers in
Plant science 3, article 309. - Clough, S. J. and Bent A. F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743.
- Deprez, T., Vernhettes, S., Fagard, M., Refregier, G., Desnos, T., Aletti, E., Py, S and Höfte, H. 2002. Resistance against herbicide Isoxaben and cellulose deficiency caused by distinct mutations in same cellulose synthase isoform CESA6. Plant Physiol. 128, 482-490.
- Endler, A. and Persson, S. 2011. Cellulose synthases and synthesis in Arabidopsis. Mol. Plant 4, 199-211.
- Festucci-Buselli, R. A., Otoni, W. C. and Joshi, C. P. 2007. Structure, organization and functions of cellulose synthase complexes in higher plants. Braz. J. Plant. Physiol. 19, 1-13.
- Garcia-Angulo, P., Alonso-Simon, A., Encina, A., Alvarez, J. M., and Acebes, J. L. 2012. Cellulose biosynthesis inhibitors: comparative effect on bean cell cultures. Int J Mol Sci 13, 3685-3702.
- Harris, D. M., Corbin, K., Wang, T., Gutierrez, R., Bertolo, A. L., Petti, C., Smilgies, D. M., Estevez, J. M., Bonetta, D., Urbanowicz, B. R., Ehrhardt, D. W., Somerville, C. R., Rose, J. K., Hong, M., and Debolt, S. 2012. Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase. Proc Natl Acad Sci USA 109, 4098-4103.
- Heim, D. R., Roberts, J. L., Pike, P. D. and Larrinua, I. M. 1989. Mutations of a locus of Arabidopsis thaliana confers resistance to the herbicide isoxaben. Plant Physiol. 90, 146-150
- Kimura, S., Laosinchai, W., Itoh, T., Cui, X., Linder, R. and Brwon, R. M. Jr. 1999. Immunogold labeling of rosette terminal cellulose synthesizing complexes in the vascular plant Vigna angularis. The Plant Cell 11, 2075-2085.
- Lei, L., Li, S. and Gu, Y. 2012. Cellulose synthase complexes: composition and regulation. Frontiers in plant science, 3, article 75.
- Murashige. T, and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant 15, 473-497.
- Scheible, W. R., Eshed, R., Richmond, T., Delmer, D. and Somerville, C. 2001. Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidospsis Ixr1 mutants. Proc. Nat. Acad. Sci. USA 98, 10079-10084.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013142968A1 (en) | 2012-03-26 | 2013-10-03 | Governing Council Of The University Of Toronto | Compositions, methods, and plant genes for the improved production of fermentable sugars for biofuel production |
WO2015162143A1 (en) | 2014-04-23 | 2015-10-29 | Basf Se | Plants having increased tolerance to herbicides |
Family Cites Families (30)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2011914A (en) | 1928-06-29 | 1935-08-20 | Du Pont | Fibrous material and process of producing it |
KR100386337B1 (en) | 1993-12-09 | 2004-03-24 | 토마스 제퍼슨 대학교 | Compounds and Method for Site-Specific Mutations in Eukaryotic Cells |
US6140466A (en) | 1994-01-18 | 2000-10-31 | The Scripps Research Institute | Zinc finger protein derivatives and methods therefor |
WO1995019431A1 (en) | 1994-01-18 | 1995-07-20 | The Scripps Research Institute | Zinc finger protein derivatives and methods therefor |
GB9824544D0 (en) | 1998-11-09 | 1999-01-06 | Medical Res Council | Screening system |
USRE45721E1 (en) | 1994-08-20 | 2015-10-06 | Gendaq, Ltd. | Relating to binding proteins for recognition of DNA |
US5789538A (en) | 1995-02-03 | 1998-08-04 | Massachusetts Institute Of Technology | Zinc finger proteins with high affinity new DNA binding specificities |
US5760012A (en) | 1996-05-01 | 1998-06-02 | Thomas Jefferson University | Methods and compounds for curing diseases caused by mutations |
US5731181A (en) | 1996-06-17 | 1998-03-24 | Thomas Jefferson University | Chimeric mutational vectors having non-natural nucleotides |
US5925523A (en) | 1996-08-23 | 1999-07-20 | President & Fellows Of Harvard College | Intraction trap assay, reagents and uses thereof |
GB9710807D0 (en) | 1997-05-23 | 1997-07-23 | Medical Res Council | Nucleic acid binding proteins |
GB9710809D0 (en) | 1997-05-23 | 1997-07-23 | Medical Res Council | Nucleic acid binding proteins |
US6140081A (en) | 1998-10-16 | 2000-10-31 | The Scripps Research Institute | Zinc finger binding domains for GNN |
US6453242B1 (en) | 1999-01-12 | 2002-09-17 | Sangamo Biosciences, Inc. | Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites |
US6534261B1 (en) | 1999-01-12 | 2003-03-18 | Sangamo Biosciences, Inc. | Regulation of endogenous gene expression in cells using zinc finger proteins |
US6794136B1 (en) | 2000-11-20 | 2004-09-21 | Sangamo Biosciences, Inc. | Iterative optimization in the design of binding proteins |
US20020061512A1 (en) | 2000-02-18 | 2002-05-23 | Kim Jin-Soo | Zinc finger domains and methods of identifying same |
AU2001263155A1 (en) | 2000-05-16 | 2001-11-26 | Massachusetts Institute Of Technology | Methods and compositions for interaction trap assays |
JP2002060786A (en) | 2000-08-23 | 2002-02-26 | Kao Corp | Germicidal stainproofing agent for hard surface |
GB0108491D0 (en) | 2001-04-04 | 2001-05-23 | Gendaq Ltd | Engineering zinc fingers |
AU2002336373A1 (en) | 2001-08-20 | 2003-03-03 | The Scripps Research Institute | Zinc finger binding domains for cnn |
CA2474486C (en) | 2002-01-23 | 2013-05-14 | The University Of Utah Research Foundation | Targeted chromosomal mutagenesis using zinc finger nucleases |
EP2368982A3 (en) | 2002-03-21 | 2011-10-12 | Sangamo BioSciences, Inc. | Methods and compositions for using zinc finger endonucleases to enhance homologous recombination |
CA2497913C (en) | 2002-09-05 | 2014-06-03 | California Institute Of Technology | Use of chimeric nucleases to stimulate gene targeting |
US7888121B2 (en) | 2003-08-08 | 2011-02-15 | Sangamo Biosciences, Inc. | Methods and compositions for targeted cleavage and recombination |
US8409861B2 (en) | 2003-08-08 | 2013-04-02 | Sangamo Biosciences, Inc. | Targeted deletion of cellular DNA sequences |
WO2007014275A2 (en) | 2005-07-26 | 2007-02-01 | Sangamo Biosciences, Inc. | Targeted integration and expression of exogenous nucleic acid sequences |
US8399218B2 (en) | 2007-09-27 | 2013-03-19 | Dow Agrosciences, Llc | Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes |
US20110239315A1 (en) | 2009-01-12 | 2011-09-29 | Ulla Bonas | Modular dna-binding domains and methods of use |
CN103025344B (en) | 2010-05-17 | 2016-06-29 | 桑格摩生物科学股份有限公司 | Novel DNA-associated proteins and application thereof |
-
2017
- 2017-08-01 EP EP17751685.3A patent/EP3497213B1/en active Active
- 2017-08-01 JP JP2019507272A patent/JP7066126B2/en active Active
- 2017-08-01 WO PCT/EP2017/069386 patent/WO2018029034A1/en unknown
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013142968A1 (en) | 2012-03-26 | 2013-10-03 | Governing Council Of The University Of Toronto | Compositions, methods, and plant genes for the improved production of fermentable sugars for biofuel production |
WO2015162143A1 (en) | 2014-04-23 | 2015-10-29 | Basf Se | Plants having increased tolerance to herbicides |
Non-Patent Citations (5)
Title |
---|
Harris et al. (PNAS, 109-4098-4103, 2012). * |
Mizuki et al.(J Exp. Bot., 67:533-542, Jan. 2016). * |
PCT International Search Report and Written Opinion, Application No. PCT/EP2017/069386, dated Oct. 20, 2017, 10 pages. |
Tateno et al., Cellulose Biosynthesis Inhibitors—A Multifunctional Toolbox, Department of Hroticulture, Unviersity of Kentucky, Journal of Experimental Botany, vol. 67, No. 2 pp. 533-542, Nov. 19, 2015, doi:10.1093/jxb/erv489. |
Zhubing, Hu, et al., Genome Editing-Based Engineering of CES A3 Dual Cellulose-Inhibitor-Resistant Plants. (Jun. 2019) www.plantphysiol.org vol. 180, pp. 827-836. |
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